Journal
JOURNAL OF VIROLOGY
Volume 79, Issue 3, Pages 1713-1723Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.79.3.1713-1723.2005
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Funding
- NIAID NIH HHS [AI-46984] Funding Source: Medline
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Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4(D12)) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gpl20-CD4(D12) and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4(D12) to 17b is not affected by the addition of excess soluble CD4(D12), while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4(D12). Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type I isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4(D12). These antisera were depleted of anti-CD4(D12) antibodies by being passed over a column containing immobilized CD4(D12). The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4(D12) competed with the CD4(i) antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4(i) epitope were generated by the gp120-CD4(D12) immunogen, these antibodies were nonneutralizing.
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